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Miniaturisation des technologies d’imagerie de fluorescence pour assister la chirurgie mini-invasive
Paul Dorval
To cite this version:
Paul Dorval. Miniaturisation des technologies d’imagerie de fluorescence pour assister la chirurgie mini-invasive. Traitement du signal et de l’image [eess.SP]. Université de Strasbourg, 2015. Français.
�NNT : 2015STRAD005�. �tel-01477107�
UNIVERSITÉ DE STRASBOURG
ÉCOLE DOCTORALE MSII
ICube UMR 7357 – Laboratoire des sciences de l’ingénieur, de l’informatique et de l’imagerie
THÈSE
présentée par :Paul Dorval
soutenue le : 25 Février 2015
pour obtenir le grade de :
Docteur de l’ Université de Strasbourg
Discipline/ Spécialité
: Signal, Image, Automatique, Robotique (SIAR) - Image et vision
Miniaturisation des technologies d’imagerie de fluorescence pour assister la chirurgie mini-invasive
THÈSE dirigée par :
M. POULET Patrick Maître de Conférences-Praticien Hospitalier, Université de Strasbourg
RAPPORTEURS :
M. BOCCARA Claude Professeur des Universités, ESPCI ParisTech M. MORDON Serge Directeur de recherche, INSERM
AUTRES MEMBRES DU JURY :
M. GAYET Brice Professeur des Universités-Praticien Hospitalier, Université Paris Descartes et Institut Mutualiste Montsouris
M. SOLER Luc Professeur des Universités-Praticien Hospitalier, Université de Strasbourg
M. UHRING Wilfried Professeur des Universités, Université de Strasbourg
Remerciements
Le dispositif CIFRE a pour but de sortir le travail de th`ese du laboratoire et de cr´eer une synergie entre centres de recherches acad´emiques et entreprises inno- vantes. Ce travail de th`ese a ´et´e majoritairement effectu´e au sein de l’entreprise Fluoptics `a Grenoble et c’est tout naturellement que mes premiers remerciements se tournent vers Odile Allard et Philippe Rizo pour la confiance et l’opportunit´e qu’ils m’ont offert de conduire ce projet. J’exprime tout particuli`erement ma gratitude envers Philippe qui a su, tout au long de la th`ese, motiver et suivre d’un regard bienveillant le travail accompli.
Je remercie aussi sinc`erement Patrick Poulet d’avoir accept´e de diriger ce travail et d’avoir partag´e avec moi son expertise sur le domaine. Je remercie aussi le laboratoire iCube de Strasbourg pour m’avoir accueilli parmi leurs doctorants.
Je tiens `a remercier Claude Boccara et Serge Mordon d’avoir accept´e de rap- porter ce travail. Je remercie ´egalement les autres membres du jury, Brice Gayet, Luc Soler et Wilfried Uhring.
Ma reconnaissance va aussi vers les personnes ext´erieures ayant particip´e `a ce travail : Fabien Stenard, Gabriele Barabino, Alexandre Filippello, Christian Righini, Ihab Atallah et Jean-Luc Coll notamment pour la validation preclin- ique et clinique des instruments ; Jean-Marc Dinten, Michel Berger, Patricia Le Coupanec et Charlotte Emain en ce qui concerne les collaborations avec le CEA- LETI.
Alors bien sˆur, je remercie chaleureusement mes coll`egues de Fluoptics. En pre- mier lieu Norman avec qui j’ai ´enorm´ement appris, puis St´ephanie, Pascal, Yoann, Diane, Anthony, Mathilde, Muriel, Oph´elie et tous les autres qui ont permis de rendre ces presque 4 ann´ees totalement uniques.
Enfin, je remercie ma famille, mes amis et Laurianne, sans qui tout ¸ca n’aurait pas ´et´e possible.
The miniaturization of fluorescence image-guided
technologies to assist minimally invasive surgery
Contents
Remerciements 2
Contents . . . 3
List of Figures . . . 5
List of Tables . . . 8
Glossary 11 Introduction 13 1 Fluorescence Image-Guided Surgery 17 1.1 Introduction . . . 19
1.2 Fluorescence imaging . . . 20
1.2.1 Light and living tissues . . . 20
1.2.2 Fluorescence principle and imaging modality . . . 24
1.2.3 Why fluorescence image-guided surgery ? . . . 29
1.3 Fluorescence image-guided surgery, a survey . . . 30
1.3.1 Systems . . . 30
1.3.2 Indications . . . 34
1.4 Conclusion of the chapter . . . 41
Bibliography . . . 41
2 The FluostickTM, a miniaturized hand-held fluorescence image- guided surgery device 47 2.1 Introduction . . . 49
2.2 The miniaturization of existing technologies . . . 50
2.2.1 Purpose of the development . . . 50
2.2.2 Camera . . . 51
2.2.3 Optics, Filtering and Excitation . . . 53
2.2.4 Ergonomics and conclusion on miniaturization purpose . . 56
2.3 Evaluation of the system . . . 57
2.3.1 Imaging and Fluorescence performance . . . 57
2.3.2 Preclinical evaluation . . . 60
2.3.3 Clinical evaluation . . . 62
2.4 Conclusion of the chapter . . . 67
Bibliography . . . 67
3.1 Introduction . . . 74
3.2 The purpose of the development and the particularities of mini- invasive procedures . . . 75
3.2.1 The purpose of the development . . . 75
3.2.2 Minimally invasive surgery . . . 75
3.2.3 Translation from open surgery to mini-invasive surgery . . 76
3.2.4 Fluorescence imaging and digestive coelioscopy . . . 83
3.2.5 Fluorescence and minimally invasive surgery, existing in- strumentation . . . 85
3.3 The technical development of the FluoMISTM . . . 89
3.3.1 Build on existing material . . . 89
3.3.2 Specific development for the FluoMISTM . . . 90
3.3.3 Final specifications . . . 97
3.4 Evaluation of the system . . . 101
3.4.1 Performances . . . 101
3.4.2 In-vivo evaluation . . . 101
3.5 Conclusion of the chapter . . . 108
Bibliography . . . 108
4 Improve the FluoMISTM, breakthrough in the technology and prospective studies 113 4.1 Introduction . . . 115
4.2 The development of a single imager fluorescence imaging device for mini-invasive surgery . . . 116
4.2.1 Principle and purpose of the development . . . 116
4.2.2 System architecture . . . 117
4.2.3 Camera and sensor evaluation . . . 119
4.2.4 Sequence and image acquisition . . . 122
4.2.5 Filtering . . . 124
4.2.6 Mechanics . . . 125
4.3 System evaluation, comparison with the FluoMISTM system . . . 126
4.3.1 Color image . . . 126
4.3.2 Depth of field . . . 127
4.3.3 Fluorescence sensitivity, comparison with previous devel- opment . . . 129
4.4 Distal sensor and perspective studies . . . 131
4.4.1 In-vivo evaluation . . . 131
4.4.2 Distal sensor . . . 131
4.5 Conclusion of the chapter . . . 134
Bibliography . . . 134
Conclusion 137 A Image sensor characterization 141 A.1 Imaging technologies . . . 141
A.1.1 CCD sensor . . . 141
A.2 Characteristic values of an image sensor . . . 149
A.3 Characterization methodology . . . 151
A.3.1 Experimental setup . . . 151
A.3.2 Quantization step . . . 152
A.3.3 Noise . . . 154
A.3.4 Linearity . . . 157
A.3.5 Photo Response Non-Uniformity . . . 157
A.3.6 Quantum Yield . . . 158
A.4 Sample results of evaluations . . . 159
B Optics characterization tools 163 B.1 ICG limit of detection : drops test . . . 163
B.2 Resolution determination . . . 164
B.3 Depth of field . . . 166
B.3.1 Hyperfocal . . . 166
B.3.2 Depth of field, d≫f . . . 166
B.3.3 Depth of field, d≈f . . . 167
B.3.4 Discussion and application . . . 167
Communications 169 R´esum´e 171
List of Figures
1.1 Electromagnetic spectra . . . 201.2 Absorption theory . . . 21
1.3 Scattering theory . . . 22
1.6 Bruker preclinical . . . 26
1.7 Fluorescence imaging principle . . . 27
1.8 Methylene blue spectra . . . 28
1.9 Indocyanine Green spectra . . . 28
1.10 Why fluorescence image-guided surgery . . . 29
1.11 Systems survey, arm-held type . . . 32
1.12 Systems survey hand-held type . . . 33
1.13 PDE . . . 35
1.14 FLARE . . . 35
1.15 Fluobeam . . . 36
1.16 Flap surgery with PDE . . . 37
1.17 Hepatic surgery with PDE . . . 38
1.18 Lymphoedema surgery with Fluobeam . . . 39
1.19 Graft assessment with Hypereye . . . 40
1.20 CABG with Spy . . . 40
2.1 The FluostickTMFIGS System . . . 51
2.2 Layout of the optical head . . . 53
2.3 Mechanical integration . . . 53
2.4 Filtering . . . 55
2.5 Shape of the optical head . . . 57
2.6 Drops of ICG . . . 58
2.7 Signal to Noise Ratio . . . 59
2.8 Resolution chart . . . 60
2.9 Preclinical samples with the FluostickTM . . . 61
2.10 Preclinical samples with the FluobeamR . . . 61
2.11 Preclinical samples with the FluostickTM2 . . . 62
2.12 Liver tumor . . . 63
2.13 Picture from the clinical trial . . . 64
2.14 Picture from the clinical trial 2 . . . 65
2.15 Picture from the clinical trial 3 . . . 66
3.1 Rod Lens System, description . . . 76
3.2 Laparoscope characterization . . . 77
3.3 Laparoscope characterization 3 . . . 79
3.4 Result of the laparoscopes comparison . . . 80
3.5 Survey FIGS systems for open surgery . . . 82
3.6 Olympus Endoeye HD . . . 82
3.7 Gallbladder and cystic canal configuration . . . 84
3.8 Samples acquired with the Olympus FIGS system . . . 86
3.9 Samples acquired with the Pinpoint system . . . 87
3.10 Samples acquired with the DaVinci FIGS system . . . 88
3.11 Sample acquired with the Flare system . . . 88
3.12 Camera selected for the fluorescence channel . . . 89
3.13 Mechanical integration . . . 91
3.14 Inner architecture of the system . . . 92
3.15 Dichroic filter . . . 93
3.16 Schott Led Engine . . . 94
3.17 Generation of white and excitation light . . . 95
3.18 Light box filters . . . 96
3.19 Dichroic filter . . . 96
3.20 Inner architecture of the light box . . . 97
3.21 White light spectrum . . . 98
3.22 Definition of the Laser class . . . 99
3.23 Resolution target . . . 100
3.24 Drops of ICG . . . 101
3.25 Preclinical evaluation . . . 102
3.26 Preclinical evaluation 2 . . . 103
3.27 Preclinical evaluation 3 . . . 104
3.28 Preclinical evaluation 4 . . . 105
3.29 Preclinical evaluation 5 . . . 107
3.30 Preclinical evaluation 6 . . . 107
4.1 Principle of the system . . . 117
4.2 Architecture of the system . . . 118
4.3 Boxes of the system . . . 119
4.4 Camera . . . 120
4.5 CMV2000 . . . 121
4.6 Samples acquired with the system . . . 122
4.7 Sequence of acquisition . . . 123
4.8 Custom notch filter . . . 124
4.9 Custom notch filter 2 . . . 125
4.10 Head of the system . . . 125
4.11 Systems comparison, color . . . 127
4.14 Systems comparison, drops test . . . 131
4.15 Systems architecture . . . 132
4.16 System architecture, distal sensor . . . 133
A.1 MOS capacity, photoelectric effect in a photo-diode . . . 142
A.2 CCD’s photo-site architecture . . . 143
A.3 Diagram of a Full-Frame CCD sensor . . . 144
A.4 Diagram of a Block-Transfer CCD sensor . . . 145
A.5 Diagram of an Interline CCD sensor . . . 146
A.6 Examples of glare artifacts . . . 147
A.7 Several windowing achievable on CMOS and CCD sensor . . . 147
A.8 Diagram of a CMOS sensor . . . 148
A.9 CMOS’ photo-gate architecture . . . 149
A.10 CMOS’ photo-diode architecture . . . 149
A.11 Sensor global response, noise evolution, EMVA type . . . 151
A.12 Diagram of the measurement setup . . . 152
A.13 Curve quantization step . . . 153
A.14 Curve readout noise and dark current . . . 155
A.15 Curve dark current and offset . . . 156
A.16 Curve linearity . . . 157
A.17 Curve quantum yield . . . 159
A.18 Quantum yield comparison . . . 161
B.1 Drops of ICG . . . 164
B.2 Resolution chart . . . 165
B.3 Line pairs / mm in USAF resolving power test target 1951 . . . . 165
List of Tables
1.1 Preclinical systems . . . 251.2 systems . . . 31
1.3 systems comparison . . . 34
2.1 Features and miniaturization . . . 57
2.2 Specifications . . . 60
3.1 Laparoscope characterization 2 . . . 78
3.2 Comparison between open and mini-invasive surgery . . . 80
4.1 Camera characteristics . . . 120
4.2 Evaluation of the depth of field, system comparison . . . 129
A.1 Cameras comparison . . . 160
Glossary
CABG: Coronary Artery Bypass Grafting CCD: Charged-Coupled Devicee
CEA: Comissariat `a l’Energie Atomique
CMOS: Complementary Metal Oxide Semi-Conductor
CMOS-APS: Complementary Metal Oxide Semi-Conductor Active Pixel Sensors CNRS: Centre National de la Recherche Scientifique
CRI: Color-Rendering Index CE: Conforme aux Egixences
FDA: Federal Drugs Administration FIGS: Fluorescence Image-Guided Surgery FPS: Frame Per Second
ICG: IndoCyanine Green
INSA: Institut National des Sciences Appliqu`ees
Laser: Light Amplification by Stimulated Emission of Radiation LED: Light-Emitting Diode
HNSCC: Head and Neck Squamous Cell Carcinoma MPE: Maximum Permissible Exposure
NIR: Near Infra-Red NUV: Near Ultra-Violet OD: Optical Density
PCB: Printed Circuit Board ROI: Region Of Interest SLN: Sentinel Lymph Node
SLNB: Sentinel Lymph Node Biopsy SNR: Signal-to-Noise Ratio
USAF1951: United-States Air Force 1951 USB: Universal Serial Bus
Introduction
In the last 10 years, significant improvements have been achieved in all aspects of medical imaging. As a way to create visual representation of the surface or interior of the body or specific organs, medical imaging is a powerful tool to help the physician in his work. In surgery, it gives clues to diagnose disease and guides complex procedures.
Fluorescence image-guided surgery is a particular optical imaging modality. It exploits the properties of light sources in order to image anatomic particularities of tissues thanks to contrast agents, or dyes, previously injected to the patient.
This technique gives information in real-time during open surgery to the physi- cian and help him in performing his procedure.
The modality has shown a huge potential in oncology, vascular and lymphatic related surgeries. To be more specific, fluorescence is useful for sentinel lymph node mapping or biopsy in oncologic procedures (breast, skin, gastric and col- orectal cancers), perfusion visualization of free flaps in reconstructive surgery, tumor resection and general vascular and lymphatic mapping.
Besides, since 1983 and the first laparoscopic appendectomy, minimally inva- sive surgical techniques does not cease to develop. The advantages of the method are numerous. First of all, it is noticed a decrease operative trauma with a lim- ited blood loss during surgery. So even though the surgery might take longer in comparison to open procedures, the hospitalization time is always much shorter.
With less pain and scarring, the patient bears less post-surgical complications.
Today, the majority of fluorescence image-guided surgery indications are related to open surgery procedures, but the potential of the technology for mini-invasive surgeries is tremendous. Indeed, one of the particularities of mini-invasive surgery is the fact that the surgeon can only rely on the image displayed on the screen and cannot feel the tissue structure as he would do by touching them in an open- surgery procedure. Therefore, complementary information to the anatomical color image is going to become mandatory and NIR fluorescence image-guided
common mini-invasive surgeries. In these cases, fluorescence imaging is used to image distinctive anatomical part such biliary vessels in the case of cholecystec- tomy or tumor margins and vascularization for the nephrectomy. Nevertheless, few devices are commercially available for such procedures and the technology is not widely developed in this very demanding field. The direct translation from open to mini-invasive surgeries for the existing fluorescence image-guided devices is not trivial due to their size and overall performance.
Although the potential of fluorescence imaging and mini-invasive procedures seemed promising, challenges have been identified in the translation of the tech- nology from open to mini-invasive procedures. The purpose of the thesis was to first identified the milestones and then addressed them in order to develop a reli- able fluorescence image-guided device for mini-invasive surgeries. The obstacles identified to the development were mainly a size issue and a sensitivity problem due to the use of complex optical elements and small image sensors.
This thesis will focus on several subjects. First, the color channel and the way to overlaid the color image to the fluorescence information. Several solution will be presented and a one sensor architecture and a two sensors device will be de- veloped an compared.
A breakthrough in the size of image sensor used will also be exposed. In fact, large scientific image sensor, commonly used in NIR fluorescence imaging, re- quired too much space and cannot be integrated in mini-invasive camera.
The depth of field of the imaging system will also be a problem assessed in the thesis. In general, ergonomics of the fluorescence image-guided devices will be discussed.
Finally, one of the big challenge identified is the need of high framerate acquisi- tion. Because the imaging device has to provide real-time images to the surgeon, the framerate of the system must exceed 25f ps. Moreover, in the case of a single sensor system, a pulsed acquisition mode is envisaged. In this case the system should be able to acquire images at 50f psor more. High framerates induce short exposure time and that is, a priori, inadequate with the low fluorescence light emission of the probes imaged.
To solve and progressively address the several issues identified above, the thesis will present the development of three distinctive devices. A first step of miniaturization of the fluorescence image-guided technology for open surgery had been identified as a requirement for further mini-invasive investigation. Based
on this first step, the second development of the thesis is a 2-sensors camera device dedicated to mini-invasive procedures. Then an innovative system able to acquire color images and fluorescence images in the same time with one sensor will be presented.
The chapter 1 of the thesis will present generalities about imaging and especially fluorescence imaging. A survey of existing fluorescence image-guided surgery devices will be exposed. This chapter will clearly set the need for further inves- tigation and the purpose of the developments achieved for this thesis.
The chapter 2 will expose the development of a miniaturized device dedicated to open-surgery and specifically laparotomy, the FluostickTM. The complete de- velopment process will be exposed, as preclinical and clinical evaluations. This development will be a first step, prior of the development of mini-invasive de- vices.
The chapter 3 will present the development of a 2-sensors fluorescence imag- ing system for mini-invasive purpose called the FluoMISTM and directly derived from the development exposed in chapter 2. Characteristics and particularities of mini-invasive modality for fluorescence NIR imaging will be evaluated. Pre- clinical tests performed with the system will be balanced.
The chapter 4 will introduce a new mini-invasive single sensor fluorescence imag- ing device development. The goal of this development is to correct the drawbacks of the system presented in chapter 3. Also, it will be exposed innovative achieve- ments as the use of a sequential pulsed acquisition mode.
The conclusion will expose the achievements of the thesis. Also, a discussion will be conducted about the different architectures proposed for a mini-invasive FIGS system. Perspective work and potential studies will end the conclusion.
Appendix will describe characterizations tools involved in the thesis. An image sensor test method will be exposed, as sample results of camera evaluations. A ICG limit of detection determination will be presented and clues about the depth of field and the resolution determination will be given.
Chapter 1
Fluorescence Image-Guided Surgery
What you will find in this chapter:
This chapter is a survey of the specific technology of fluorescence image-guided surgery. It will be exposed the principle of fluorescence, existing fluorescence image-guided surgery systems and major addressed indications. The chapter clearly sets the need for further investigation and the purpose of the developments achieved for this thesis.
Contents
1.1 Introduction . . . 19
1.2 Fluorescence imaging . . . 20
1.2.1 Light and living tissues . . . 20
1.2.2 Fluorescence principle and imaging modality . . . 24
1.2.3 Why fluorescence image-guided surgery ? . . . 29
1.3 Fluorescence image-guided surgery, a survey . . . 30
1.3.1 Systems . . . 30
1.3.2 Indications . . . 34
1.4 Conclusion of the chapter . . . 41
Bibliography . . . 41
Figures
1.1 Electromagnetic spectra . . . 201.2 Absorption theory . . . 21
1.3 Scattering theory . . . 22 1.4 Tissues absorption . . . 24 1.5 Jablonski diagram . . . 25 1.6 Bruker preclinical . . . 26 1.7 Fluorescence imaging principle . . . 27 1.8 Methylene blue spectra . . . 28 1.9 Indocyanine Green spectra . . . 28 1.10 Why fluorescence image-guided surgery . . . 29 1.11 Systems survey, arm-held type . . . 32 1.12 Systems survey hand-held type . . . 33 1.13 PDE . . . 35 1.14 FLARE . . . 35 1.15 Fluobeam . . . 36 1.16 Flap surgery with PDE . . . 37 1.17 Hepatic surgery with PDE . . . 38 1.18 Lymphoedema surgery with Fluobeam . . . 39 1.19 Graft assessment with Hypereye . . . 40 1.20 CABG with Spy . . . 40
Tables
1.1 Preclinical systems . . . 25 1.2 systems . . . 31 1.3 systems comparison . . . 34
CHAPTER 1. FLUORESCENCE IMAGE-GUIDED SURGERY
1.1 Introduction
In recent years, a lot of improvements have been achieved in all aspects of medical imaging. This chapter will present in details the specific technology of fluorescence imaging. Numerous reviews of the technique have been published [1, 2, 3, 4, 5, 6, 7, 8] and laboratories and companies are working on the subject worldwide. The modality has shown a huge potential in oncology, vascular and lymphatic related surgeries. To be more specific, fluorescence is useful for sentinel lymph node in oncologic procedures (breast, skin, gastric and colorectal cancers in majority, [9, 10, 11, 12, 13, 14, 15, 16, 17, 18]), vascular visualization of free flaps in reconstructive surgery, [19], tumor resection, [20, 21] and general vascu- lar, [22, 23, 24, 25], and lymphatic mapping, [26, 27]. The goal of Fluorescence Image-Guided Surgery (FIGS) systems is to image fluorescent contrast agents, also called probes, previously injected to the patient. To achieve this, the system is able to send specific excitation light absorbed by the probes to be imaged and to collect the subsequent light emitted. Indocyanine Green (ICG) and methylene blue are FDA-approved probes for fluorescence procedures, [28, 29, 30, 31, 32].
Other contrast agents such as Patent Blue V, approved in Europe, have shown a potential for fluorescence image-guided surgeries, [16, 33]. Targeted contrast agents are also under development. They are mostly developed to be tumor specific, [34, 35, 36, 37, 38]. These most common probes emits light in a near infrared (NIR) spectrum region comprised between 650 and 900nm.
The FIGS system mostly consists in an imaging head linked to one or several control boxes including hardware and software to acquire and display images.
Due to their size and weight, most of the available systems on the market are fixed to a mechanical arm and are able to image the surgical field from the top only. The system is built around an imaging sensor, which is, thanks to filters, able to collect specifically the fluorescence light emitted by the excited probes, [39, 40, 41]. The ambient light of the theater, as well as the shadowless surgical light, contain NIR light. It’s particularly true for xenon or tungsten type light.
Therefore, to perform good fluorescence acquisitions, the light of the operating room must be turned off. Some FIGS systems provide an additional filtered white light which help the surgeon to perform his procedures but will not disrupt the acquisition of fluorescence emission light.
1.2 Fluorescence imaging
1.2.1 Light and living tissues
Medical and optical imaging
Medical imaging, as the process to create visual representation of the body for clinical analysis and medical intervention, is divided into categories according to the electromagnetic spectrum. The figure 1.1 shows the electromagnetic spec- trum and names different areas. Medical imaging applications use most of the domains of this spectrum. For instance, radiology deals with the X-rays part of the spectrum, Single Photon Emission Computed Tomography (SPECT) and Positron Emission Tomography (PET) deals with the Gamma-rays part of the spectrum, and Magnetic Resonance Imaging (MRI) with the radio-frequency range.
Fluorescence image-guided surgery, the thematic of this thesis, is part of Optical imaging. Optical imaging modalities focus on the visible part of the spectrum extended to the near-ultraviolet (NUV) and near-infrared areas (NIR).
Figure 1.1: Electromagnetic and light spectra
Of course, the first and main in-vivo optical imaging method involve only the eye of the surgeon who is able to distinguish anatomical structures, color and aspects of the tissues and organs thanks to an adequate surgical shadow-less
CHAPTER 1. FLUORESCENCE IMAGE-GUIDED SURGERY
light and a direct visualization. Nonetheless, the use of special sensors, cameras and lights can bring the surgeon a further level of information.
Light interactions with the tissues
At a microscopic level, two phenomenons describe the light interaction with molecules: the absorption and the scattering. The absorption coefficient,µa, of a medium expresses its disposition to block incoming light. In fact, it also expresses how the energy of incoming photons will be taken up by the molecules of the medium considered. The coefficient µa is expressed in cm−1 and it described by the following equation:
µa =ρaσa (1.1)
The coefficient ρa, expressed in cm−3, is the concentration of molecules in the medium considered. The parameter σa is called effective cross-section of a molecule and it expressed in cm2. The cross section of a molecule is an ex- pression of the theoretical shadow created by the molecule exposed to a beam of light. The following figure, 1.2, is a schematic of the cross-section concept. The molecule is represented by a sphere with a geometrical cross-sectionA expressed in cm2, the absorption efficiency coefficient, Qa is dimensionless.
Figure 1.2: Absorption schematic, from [42]
From this absorption phenomenon resulted the Beer-Lambert law which ex- pressed the attenuation of a collimated beam light crossing a non-scattering medium:
I(z) = I0e−µaz (1.2) The coefficient I0 is initial intensity of the beam light, I is the intensity at a distance z in the medium expressed in cm.
In order to describe the light interaction with tissues, the absorption is not the only coefficient to take into account. The other phenomenon that will be de- scribed here is called the scattering effect. Similarly to the absorption coefficient µa, the scattering coefficient µs expresses the disposition of a medium to scatter incoming light. The coefficient µs is expressed in cm−1 and it described by the following equation:
µs =ρsσs (1.3)
The coefficient ρs, expressed in cm−3, is the concentration of molecules in the medium considered. The parameterσsis called effective cross-section of a molecule and it expressed in cm2. The following figure, 1.3, is a schematic of the cross- section concept. The molecule is represented by a sphere with a geometrical cross-section A expressed in cm2, the scattering efficiency coefficient, Qs is di- mensionless.
Figure 1.3: Scattering schematic, from [42]
The path of light in a scattering medium is not only described by the scatter- ing coefficientµs. Indeed, a scattering event changes the direction by a scattering angle θ. The angular distribution of scattered photons as a function of the angle θ is called the phase function, f(θ). The phase function depends on the size of the scattering particles. For biological media, it can be expressed by the empiric Henyey-Greenstein law, [43]:
f(θ) = 1 4π
1−g2
(1 +g2+ 2gcosθ)3/2 (1.4)
In this equation, g is called the anisotropy factor, defined as the mean value of the cosine of scattering angles, g =< cosθ >. Finally, the scattering properties of
CHAPTER 1. FLUORESCENCE IMAGE-GUIDED SURGERY
a medium can be expressed by 2 parameters, the scattering coefficientµsand the anisotropy factor g. When the medium is highly scattering, these 2 parameters could be expressed by the reduced scattering coefficient µ′s:
µ′s = (1−g)µs (1.5)
This coefficient is the opposite of the mean path-length of the light, the limit after that the beam will loose ballistic information.
As a function of its wavelength, incoming light will interact differently with the tissue. The two aspects, important to consider when talking about optical imaging, are the absorption and the reduced scattering coefficients of the tis- sues. The figure 1.4 gives the absorbance of various tissue and blood components from 100nm to 10000nm. In living issue, the major absorbers are water, lipids, oxyhemoglobin and deoxyhemoglobin. When the absorbance factor is low, the light will be able to penetrate deeper in the tissues, but encountering multiple scattering and following a diffuse transport.
Optical imaging for diagnostic and therapeutic applications in biology and medecine is more efficient in a range spreading from 600nm to 1000nm, where the ab- sorbance coefficient of the tissues is lowest and scattering lower than in the vis- ible range. In fact, it is usually assumed that the reduced scattering coefficient µs depends on the wavelength according to a power law:
µ′s =a.λ−bwith b between 0.8 and 1.3, see [44, 45] (1.6) The window between 600nmand 1000nmis called the imaging or therapeutic window, [44, 46]. It represents the range where the tissue penetration is greatest.
In this window, the absorption coefficient of living tissues are around 0.1cm−1, corresponding to a path-length of 10cm before absorption. The reduced scat- tering coefficient is of the order of 10cm−1, corresponding to a mean transport length of 1mm. Any photon detected after some millimeters of propagation in a tissue encountered many scattering events. Its trajectory is much longer than the straight way, the ratio between the 2 being called the Differential Path-length Factor (DPF), ranging around 10. The penetration depth of optical methods are therefore limited to several centimeters in the NIR range, limiting the optical imaging methods to the examination of small organs or to superficial layers of bigger organs. For fluorescence imaging, we can estimate that the penetration depth is divided by ten, as compared to optical methods detecting scattered pho- tons, giving penetration depth as low as 100µm in the visible range and up to 3mm in the NIR area of the spectrum.
Figure 1.4: Absorption coefficients, from [35]
1.2.2 Fluorescence principle and imaging modality
This thesis will focus on a specific modality of optical imaging called fluorescence imaging. A substance or molecule is called fluorescent when it emits light after it has absorbed light of shorter wavelength. The principle of fluorescence could be explained by a simplified Jablonski diagram, see figure 1.5. A fluorescent molecule absorbs light with specific energy. This energy will set the molecule in an excited state. Then the molecule will try to relax and reach back its funda- mental state. By relaxing, the molecule will lose energy. This energy could be expressed by heat and light radiation.
Because the energy of light photons is directly linked to the wavelength through the relation E = hν = hcλ, where h is Planck’s constant, ν the frequency, c the celerity and λ the wavelength, the light emitted by a fluorescent molecule has a higher wavelength than the excitation incoming light. The excitation and the emission wavelengths are not unique and both are distributed along the spec- trum. This is explained by the numerous excited states accessible and the various non-radiative relaxation pathways that occur before the radiative emission of flu- orescence light. The fluorescence characteristics of a molecule can be described in a spectrum where absorption/excitation and emission are plotted in function
CHAPTER 1. FLUORESCENCE IMAGE-GUIDED SURGERY
of the wavelength and according to a relative absorption affinity and intensity of emission. Common molecules’ spectrum will be presented later in this chapter.
Figure 1.5: Jablonski diagram
Fluorescence in-vivo imaging is a widely develop technique for preclinical in- vestigation purposes. Numerous imaging systems for small animals are available on the market. The table 1.1 presents the main actors on the market. Those systems are mostly designed to image mice or other small animals. They are able to send multiple excitation wavelengths and use switching elements to adapt fil- tering to the fluorescent probes imaged. As an imaging apparatus, these devices integrate high-end scientific cooled CCD cameras.
Company System
Biospace, France Photon Imager
Berthold, Germany NightOWL and LB983 Bruker, Germany FX Pro, FX MS Pro and
Xtreme
Perkin Elmer, USA FMT1000 to FMT4000
Li-Cor Pearl Impulse
Table 1.1: The main preclinical in-vivo fluorescence imaging systems
The following figure, 1.6, is a picture of the Bruker system, FX MS Pro. The architecture of the system is common to the other existing devices. The animal is placed in a closed enclosure to perform measurements and no manipulation are allowed during the fluorescence acquisition.
Figure 1.6: The Bruker FX MS Pro, preclinical in-vivo fluorescence system Concerning medical imaging, the fluorescence could be endogenous or ex- ogenous. Endogenous fluorescence means that some tissues, due to particular molecules, demonstrate autofluorescence properties. Collagen for instance is flu- orescent under a blue or near-UV excitation. Nonetheless, most of the aut- ofluorescence of tissues is bounded to the visible part of the spectrum and few autofluorescence artifacts will occurs in the near-infrared therapeutic window de- scribed in figure 1.4.
Exogenous fluorescence imaging involves fluorescent contrast agents (also called dyes or probes). These contrast agents are molecules that can be targeted or non- targeted and mostly evolved in the visible and the NIR part of the spectrum.
The goal of Fluorescence Image-Guided Surgery (FIGS) systems is to image flu- orescent contrast agents previously injected to the patient. To achieve this, the system is able to send specific excitation light absorbed by the probes to be im- aged and to collect the subsequent light emitted. As presented before, the NIR area of the spectrum is the more adapted to imaging modalities. FIGS systems mostly deal with NIR localized contrast agents. The figure 1.7 is a schematic of a FIGS system. It mostly consists of a camera able to discriminate the fluores- cence emission from the excitation light, a light source to generate the excitation light and filters. Filters are involved to block the excitation light in front of the camera and limit the excitation light to a narrow band of the spectrum. Filters are critical components in a FIGS device. Mostly two types of filters are inte-
CHAPTER 1. FLUORESCENCE IMAGE-GUIDED SURGERY
grated in such systems. Absorptive glass filters, also called colored filters, are simple and quite cheap. It consists of a glass substrate where various inorganic or organic compounds have been added. Dichroic, or interference, filters are more complex components. They use complex interferential coating on glass or plastic substrate and are usually more selective than colored filters.
Figure 1.7: Fluorescence imaging principle, FIGS system
The principal approved probes for clinical fluorescence procedures are the Indocyanine Green (ICG), the Methylene blue and the Fluorescein. The ICG and the Methylene blue emit light in a NIR range region comprised between 650 and 900nm. The Fluorescein emits light in the green visible part of the spectrum. Figures 1.8 and 1.9 present the emission and absorption spectrum of ICG and Methylene Blue, the 2 probes which operates in NIR area of the spectrum. This thesis will mostly focus on ICG. ICG is available worldwide for intravenous injection and the main commercial name are:
• InfracyanineR from Serb laboratories, France market
• ICG-PulsionR from Pulsion, Europe market
• IC-GreenR from Akorn laboratories, USA market
• DiagnogreenR from Daiichi Sankyo, Japan market
For lymphatic related indications, which require a subcutaneous injection of ICG, its use is subject to a clinical trial submission.
Figure 1.8: On the left, the chemical structure of Methylene Blue is given. On the right, absorption and emission spectrum re displayed, figure from [47]
Figure 1.9: On the left, the chemical structure of ICG is given with key optical properties. On the right, absorption and emission spectrum are displayed for ICG diluted in phosphate-buffered saline (PBS) and fetal bovine serum (FBS), figure from [3]
CHAPTER 1. FLUORESCENCE IMAGE-GUIDED SURGERY
1.2.3 Why fluorescence image-guided surgery ?
The Methylene blue is already used as a simple colored dye in some surgeries, for instance the detection of lymph node. Thanks to its blue color, the surgeon is able to detect high concentration of the product with his eye. The same statement can be made with ICG, which is a green colored dye. It is already an attempt to what fluorescence image-guided surgery is designed for : add contrasts and information to the surgeon’s field of view. It is illustrated in figure 1.10. On the left, it is a schematic of the heart as presented in an anatomy book. On the top right, it is the real view of a heart visualized by the surgeon. The goal of FIGS is to give access to the surgeon to extra information such as the position of the vessels or improved differentiation between anatomical structures. On the bottom right, an image of a ewe’s heart has been acquired with a FIGS system after an intravenous injection of ICG. Coronary vessels are clearly identifiable.
Figure 1.10: The goal of FIGS. Left anatomical representation of the heart, top right surgeon real visualization of the heart, bottom right ewe’s heart visualiza- tion with the FluobeamR system
In the case of ICG, NIR FIGS devices have a highly improved contrast in comparison of the eye of the surgeon. What is more, they are able to detect fluorescence signal not only at the surface but few mm deeper in the tissues.
Later in this chapter, example of indications using FIGS devices and ICG will be presented.
Perspective also exists with the use of targeted contrasts agents. Some probes under development, coupled to a FIGS system, are able to target specific tumors.
For instance, it is the case with AngiostampTM developed by Fluoptics. Compa- nies as Molecular ProbesR and Perkin ElmerR provide targeted probes for the preclinical market.
1.3 Fluorescence image-guided surgery, a sur- vey
The following section will present a survey of existing Fluorescence Image-Guided Surgery (FIGS) instrumentation. Some typical characteristics of the systems will be presented and compared. Also, a non-exhaustive list of indications will be presented and commented.
1.3.1 Systems
As said before, the devices presented in this thesis mostly focus on the visualiza- tion of ICG. Nonetheless, there are not all similar and several parameters could be compared. The table 1.2 presents existing FIGS devices and gives objective information about the fluorescence excitation and emission collection, the typical working distance and field of view of the camera and the presence of an additional color image and white light. A distinction is also made concerning the clinical statue of the devices. Indeed, established company commercialize products for the medical market but laboratories also provide systems to surgeons but only in the context of clinical studies.
CHAPTER 1. FLUORESCENCE IMAGE-GUIDED SURGERY
SystemManufacturerFluorescence ExcitationFluorescence SignalWorking DistanceFieldof viewWhite lightColor + Fluo
Clinical statue Photodynamic Eye(PDER)Hamamatsu Photonics, Japan
LED805nm, 4mW/cm2>820nm20cm9.4×6.9cmnonoFDA&CEap- proved SPYRNovadaqTech- nologies,CanadaLaser806nm>835nm30cm16.7×12cmnonoFDA&CEap- proved FluobeamRFluoptics, FranceLaser750nm, 6mW/cm2780nm<X<900nm20cmfrom 20×14to 2.2×1.6cm
yesnoFDA&CEap- proved ArtemisRQuestMedi- calImaging, Netherlands
LED&Laser---yesyesCEapproved IridiumVisionsense,Is- rael----yesyesCEapproved FLARETM IsraelBethDea- conessHospital/ FrangioniLabs, USA LED745- 779nm800nm<X<848nm45cmfrom 12×9cmto 2.2×1.6cm
yesyesClinicaltrial MiniFLARETM IsraelBethDea- conessHospital/ FrangioniLabs, USA
LED745- 779nm800nm<X<848nm30cmfrom 12×9cmto 2.2×1.6cm
yesyesClinicaltrial T3-platformSurgOptix,USALaser750nm-21cmfrom1.5cm2 to107cm2yesyesClinicaltrial HyperEyeRMizuho,JapanLED760nm---yesyesClinicaltrial GXMInaviga- torInstituteofAu- tomation,ChinaLED760nm-30cm25X25cmYesYesClinicaltrial
Table 1.2: Survey of FIGS systems
In terms of ergonomics, FIGS devices could be classified into two categories.
Some of the existing systems are designed to be hand-held directly by the surgeon or by an assistant. Other devices are too heavy and bulky to do so and are integrated into mechanical arms. The figures 1.11 and 1.12 show pictures of the devices.
Figure 1.11: Images of the FIGS systems, mechanical arm-held type. A is the SPYR, B is the ArtemisR, C is the FLARETM and D is the miniFLARETM. See table 1.2 for further details
CHAPTER 1. FLUORESCENCE IMAGE-GUIDED SURGERY
Figure 1.12: Images of the FIGS systems, hand-held type. A is the PDER, B is the HypereyeR and C is the FluobeamR. See table 1.2 for further details
The main actors of the market are the PDER and the SPYR devices, the SPYR for US market and the PDER for Asia and Europe markets. The FluobeamR is the FIGS system developed by Fluoptics and it will be the basis for the developments presented in this thesis. The table 1.3 presents the results of systems comparison performed by Fluoptics. The parameters evaluated were the spatial resolution, the working distance, depth of field and the ICG limit of detection. Methods to determine the resolving power and the ICG limit of de- tection are presented in appendix B of this thesis. The gap between the limit of detection between the FluobeamR and the other devices is the more noticeable.
It is able to detect quantities of ICG 10 times lower than competitors.
System Geometric resolution
Nominal working distance
Depth of field ICG limit of detection FluobeamR 20lp/mm, i.e.
25µm
20cm 3cm, autofocus 0.5pmol
PDER 2lp/mm, i.e.
250µm
20cm 5cm, no autofocus 5pmol
SPYR 2.25lp/mm, i.e.
220µm
30cm 15cm, no autofocus 10pmol
Table 1.3: Performances comparison between the FluobeamR, PDER and SPYR
1.3.2 Indications
In the following section will be presented some applications of fluorescence image- guided surgery. This list of indications is not exhaustive and mainly focus on the use of ICG as a contrast agent. The following applications, illustrated by images from the literature acquired with the FIGS systems described in this chapter, will be presented:
• Sentinel Lymph Node mapping and biopsy
• Flap Surgery
• Hepatic Metastases
• Lymphoedema and Lymphatic mapping
• Coronary Artery Bypass Grafting Sentinel Lymph Node procedure
The Sentinel Lymph Node (SLN) procedure or Sentinel Lymph Node Biopsy (SLNB)is a common act in oncologic surgeries. The so-called sentinel lymph node is the first draining a cancer. It is postulated that it is the first place where metastasizing cancer cells would be found or detected, prior to a global dissemination. Lymph node metastasis is one of the most important signs to stage a cancer and set up an adequate therapy.
The common procedure involves the use of a radioactive colloid, the technetium- 99m. This substance, once injected near the tumor, will be rapidly fixed by the first lymph node draining the tumor. Gamma probes are used to detect the technetium and to approximately detect the position of the lymph node. The Methylene blue and the Patent blue V are commonly used in this procedure but
CHAPTER 1. FLUORESCENCE IMAGE-GUIDED SURGERY
only as a colored visual dye. They are similarly injected in the peri-tumoral area and then fixed by the sentinel lymph node. The dye helps the surgeon to find precisely the position of the node. Allergic reactions has been notified in the literature with blue dyes, [48, 49]. ICG, as a colored dye, has the advantage to be well assimilated by the patient . Nonetheless, its use in subcutaneous injection is currently only possible in the case of a clinical trial.
The exploitation of the fluorescence properties of the dyes already used in the procedure dramatically improve their contrast. Thanks to a FIGS system, the surgeon can detect very low concentration of dye even if the node is covered by few millimeters of tissue. In some particular cases, for instance the SLN procedure for breast cancer, radioactive imaging can be discarded. The node can be found only by the use of ICG and a FIGS system. Figures 1.13 and 1.14 illustrate the detection of the SLN in the case of breast cancer. The figure 1.15 is a SLNB in the case of a bladder cancer.
Figure 1.13: Sentinel Lymph Node (SLN) identification in breast cancer using the PDERsystem, [13]
Figure 1.14: SLN biopsy in breast cancer with FlareTMsystem, [17]
Figure 1.15: SLN biopsy in bladder cancer with the FluobeamR system Flap Surgery
Perforator flap surgery is widely used to reconstruct skin defects, like severe burns or excised parts like a breast after a cancer removal. The flap is a vascularized part of the body with skin and fat. It could be localized in the abdomen or lower limbs areas. Once removed, the flap is grafted to the area of the body to be reconstructed. It is important to preserve the vascularization vessels, called the perforator vessels, of the flap during resection in order to correctly perform the graft procedure.
Fluorescence imaging has shown a huge potential for this technique. Indeed, a intravenous injection of ICG and the use of a FIGS system give the surgeon a real-time angiography of the area. The surgeon can easily localizes the perforator vessels that infuse the flap and removed it properly. The figure 1.16 illustrates the dynamic localization of the perforator vessels of a flap before the resection. After an intravenous injection of ICG, the first vessels which appear at the surface of the FLAP are considered as the perforator ones.
CHAPTER 1. FLUORESCENCE IMAGE-GUIDED SURGERY
Figure 1.16: Identification of perforator vessels in the flap donor site with the PDER system, [19]
Hepatic Metastases
After an intravenous injection and a diffusion in the blood, the ICG is accumu- lated by the liver cells. In healthy liver tissue, the fluorescence ICG distribution is quite uniform on the surface visualized. Studies shown that tumors localized in the liver perturbs the ICG signal and distribution. It results a fluorescence detection and identification of the livers tumors. The fluorescence characteris- tics change with the type of tumors. For instance, in the case of hepatocellular carcinoma, ICG is strongly fixed inside the tumor. Other example, in the case a metastasis of colon cancer, the ICG is fixed at the margin of the tumor and creates a fluorescent rim around it. The figure 1.17, issued from the work of Ishizawa et al., [21], shows different fluorescent patterns around liver tumors.
Figure 1.17: Different types of hepatic tumors identified with ICG, PDERFIGS system, [21]
Lymphoedema
A lymphoedema is a partial or total obstruction of lymphatic system and chan- nels. A previous lymph node resection or the exposition to radiation during ther- apy cause such alterations. It results a lymphatic retention which could cause more severe affections and injuries. A precise identification of the lymphatic drainage of the lymphoedema is a great advantage in order to cure it. Treat- ments include non-invasive methods, such compression or exercise, and surgeries such as lymph node grafting or lymphaticovenous anastomosis.
A local injection of ICG and the use of a FIGS system is a very efficient method to map the lymphatic drainage of an area. The figure 1.18 shows an sample acquisition of the surfacic lymphatic drainage of a thigh.
CHAPTER 1. FLUORESCENCE IMAGE-GUIDED SURGERY
-
Figure 1.18: Identification of the thigh lymphatic drainage with the FluobeamR Coronary Artery Bypass Grafting
The goal of a coronary artery bypass grafting (CABG) is to improve blood flow to the heart. The procedure is needed when coronary arteries are obstructed by plaque and the supply of oxygen to the myocardium is too weak (angina pectoris). The CABG involves veins and arteries that are grafted to coronary vessels to improve the blood flow and the oxygenation of the myocardium.
Fluorescence has shown a huge potential in detecting early coronary bypass graft failures. The flow of a intravenous injection of ICG imaged by a FIGS system gives an indication about eventual leaks or abnormality in the blood flow. Figures 1.19 and 1.20 show images acquired with FIGS systems during CABG.
Figure 1.19: Graft assessment with HyperEyeRsystem, [25]
Figure 1.20: Graft assessment with SPYRsystem, [22]
BIBLIOGRAPHY
1.4 Conclusion of the chapter
This chapter presented generalities about imaging and especially fluorescence imaging. The technology of fluorescence image-guided surgery shows a huge potential in several indications, for instance in oncology, vascular and lymphatic related surgeries. Numerous devices exist and address a large scope of indications presented in this chapter. A survey of existing FIGS devices has been exposed and a comparison between the FluobeamR, developed by Fluoptics, and major actors on the fluorescence market has been presented. Nonetheless, the presented instrumentation is composed of large systems and none seemed to be directly transferable to mini-invasive surgeries. Also, as it will be presented in chapter 2, some open surgeries indications validated on preclinical models cannot be translated to clinic because of the size of the existing devices. The chapter 2 will focus on the miniaturization of a FIGS device for open-surgeries.
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